7 replies to this topic

[Michael]

Zhu X, Shen W, Wang Y, Jaiswal A, Ju Z, Sheng Q. Nicotinamide adenine dinucleotide replenishment rescues colon degeneration in aged mice. Signal Transduct Target Ther. 2017 Jul 7;2:17017. doi: 10.1038/sigtrans.2017.17. eCollection 2017. PubMed PMID: 29263919; PubMed Central PMCID: PMC5657423.
 
Compared to 3-month-old young controls, 2-year-old mice showed a spectrum of degenerative colonic phenotypes[.] ... Indeed, a significant reduction in villus number was found in the old colon versus the young colon (Figure 1c). Villus atrophy was seen in both vehicle and STC old mice (Figure 1d), while aged colon exhibited a higher frequency of hyperplasia independent of Lomotil treatment (Figure 1e). Lomotil-induced STC per se did not change the villus number or promote hyperplasia (data not shown). In addition, β-galactosidase (SA-β-gal) assay [a marker of senescent cells] revealed more positive stains in aged colon while almost none in the young colon (Figure 1f), suggesting an increased incidence of age-associated colonic senescence. This was further confirmed by decreased intestinal stem cell marker Lgr5 and increased cell senescence marker cdkn2a (p16) seen in aged colon ...
 
[Aged mice also] exhibited a significant elongated transit time and slowed stool frequency in the context of Lomotil-induced slow-transit constipation. Despite upregulated colonic tryptophan hydroxylases expression, serotonin release and expression of colon-predominant type IV serotonin receptor, reduced viability of interstitial cells of Cajal while enhanced aquaporins (Aqp1, 3 and 11) led to a less colonic motility and increased luminal dehydration in aged mice.
 
Notably, this colonic degeneration was accompanied with reduced key NAD⁺-generating enzyme expression and lowered NAD⁺/NADH ratio in aged colon.  ... Remarkably, transcription of nicotinamide mononucleotide adenylyltransferase 1, 2 and 3 (Nmnat1, 2 and 3) [convert NMN to NAD+], Nampt [converts NAM to NMN], as well as nicotinamide riboside kinase 1 and 2 (Nmrk1 and 2) [ie, the enzymes responsible for converting NR to NMN and thence NAD+] were all downregulated in old colon versus that in young colon (Figure 3a), which was further evidenced as aged colon exhibited a significantly lowered NAD⁺/NADH ratio (Figure 3b). Interestingly, almost no positive immunofluorescent staining of Nampt was seen in those hyperplastic tissues from aged colon (Figure 3c), suggesting that lowered NAD⁺ level may correlate with vulnerability of hyperplasia/tumorigenesis. ...
 
Three-month continuous administration of beta nicotinamide mononucleotide, a NAD⁺ precursor, elevated colonic NAD⁺ level and improved defecation in aged mice. [It's not clear to me whether this was "preventive" (initiated at 21 months, in time for the 24 month mark) or 24 month -MR]. ... Specifically, old mice receiving β-NMN favoured an increased colonic c-kit⁺ population (Figure 4b) and significantly improved faecal output (Figure 4c). In addition, enhanced proliferation was seen in the isolated colon epithelial cells (Figure 4d) as well as proliferating cell nuclear antigen-stained from aged mice receiving β-NMN ... In contrast, pharmacological inhibition of nicotinamide phosphoribosyltransferase, the rate-limiting enzyme for NAD⁺ biosynthesis, induced a reduction in colonic NAD content and impaired gastrointestinal function in young mice.

It's notable that they showed the rising burden of senescent cells and loss of stem cells in aged untreated colon, but don't say anything about either of these things in the aged NMN-treated animals: it would seem odd not to test this effect, and I wonder if they looked and didn't report a non-result — bad practice, but not unheard-of.

 

Although they only looked at the transcriptional level, it's notable that this is not only annother report of age-related reduction of NAMPT, but the first report AFAIK of lower NMAT and NRK transcription. If this corresponds to lower protein levels and/or activity, the molar efficacy of all NAD+ precursors would be reduced with age, which would contribute to lower NAD+ levels with age in addition to the increased consumption and also potentially be additional reasons why a somewhat higher dose might be required to achieve similar elevations.

[stefan_001]

Hi Michael,

 

Great find and evaluation. They could have indeed published more data. They also wrote this:

"In contrast, 3-month continuous administration of β-NMN, an NAD+ precursor, restored colonic NAD+ level to that of young mice (Figure 4a) and improved colon function"

 

It suggests that there was a continuous improvement over the 3 month period. If that is the case then it would require some of the transcription factors to be upregulated over time. A systemic impact.

[Michael]

Hi Michael,

Great find and evaluation. They could have indeed published more data. They also wrote this:
"In contrast, 3-month continuous administration of β-NMN, an NAD+ precursor, restored colonic NAD+ level to that of young mice (Figure 4a) and improved colon function"

It suggests that there was a continuous improvement over the 3 month period. If that is the case then it would require some of the transcription factors to be upregulated over time. A systemic impact.

I don't think we can say that it "suggests that there was a continuous improvement over the 3 month period," since they weren't measuring anything over the 3-mo period. Continuous administration does not necessarily imply continuous change in the treatment effect. I'd say that an ongoing improvement is certainly what you'd expect (it seems unlikely that the entire effect would occur instantaneously), but it could certainly be the case that much of the effect happens early with no further improvement after that, or that the treatment effect has a lag during which any treatment effect is effectuated by sheer stoichiometry rather than a change in gene expression. And either way, it wouldn't address the question of exactly the initiation of treatment (initiated at 21 months, in time for the 24 month mark, or initiated at 24 mo and measured 3 months later), nor the lack of protein or activity data, without which an effect on transcription is no more than suggestive.

I'm not clear what you;re saying when you say that a "continuous improvement ... would require some of the transcription factors to be upregulated over time." What transcription factors? Or did you just mean that transcription of the genes was progressively upregulated (or, rather, that the age-related suppression was progressively reversed). And, see again above.

No question but that there was a systemic impact — but what was the nature of that systemic impact?

[stefan_001]

Hi Michael, agree beyond the text I did not find they measured anything that would show upregulated transcription factor. I meant a systemic result of supplementation resulting in increase in NAMPT. .Here another report implicating decling NAMPT but also pointing to Nmrk1:

 

Dilated cardiomyopathy (DCM) is a severe heart disease characterized by reduced ejection fraction, altered systolic function, extracellular matrix disorganization and metabolic defects. In different mice models of DCM, the expression of the nicotinamide riboside kinase 2 (Nmrk2) implicated in the synthesis of NAD, a major coenzyme in energy metabolism and a signaling molecule, is increased. NMRK2 is similar to the muscle integrin binding protein (MIBP) that binds to the integrin α7β1 heterodimer. The role of Nmrk2 in the heart is unknown. Young Nmrk2-KO mice develop a normal cardiac hypertrophic response to angiotensin-II exposure and transverse aorta constriction (TAC) but follow-up echocardiography until 8 weeks post-TAC and during aging from 5 to 24 months revealed a more severe decrease in the EF and the development of a DCM phenotype. RT-qPCR analysis of cardiac mRNAs showed an increase in the slow, cardiac, β myosin heavy chain isoform starting at 12 months. NMRK2 was not essential to maintain myocardial NAD levels in response to pro-hypertrophic treatments and in young adults. However Nmrk1 and Nampt expression level declined strongly with aging and Nmrk2-KO mice displayed a 50% reduction in myocardial NAD levels at 24 months. The α7β1 integrin complex was repressed at this age. Immunofluorescent analyses and electron microscopy revealed a defect in laminin deposition and enlarged intercellular space in the Nmrk2-KO heart. The Nmrk2 gene is required to preserve cardiac function and structure during aging and becomes indispensable for maintaining NAD at late age. Molecular characterization of compounds modulating this pathway could give future therapeutic prospect.

http://europepmc.org...ct/med/29317476

[Michael]

Hi Michael, agree beyond the text I did not find they measured anything that would show upregulated transcription factor. I meant a systemic result of supplementation resulting in increase in NAMPT.

But then what is your basis for saying that supplementation resulted in an increase in NAMPT? They don't report any such thing, either at the protein, activity, or even the mRNA level, and there's no reason obvious to me to expect that it would do so: NAMPT's whole function is to produce NMN, and here they're bypassing its activity by supplying its product. That will raise NAD+ independently of any effect on NAMPT.
 

Here another report implicating decling NAMPT but also pointing to Nmrk1:
 
In different mice models of DCM, the expression of the nicotinamide riboside kinase 2 (Nmrk2) implicated in the synthesis of NAD,... is increased.. ...  NMRK2 was not essential to maintain myocardial NAD levels in response to pro-hypertrophic treatments and in young adults. However Nmrk1 and Nampt expression level declined strongly with aging and Nmrk2-KO mice displayed a 50% reduction in myocardial NAD levels at 24 months. The α7β1 integrin complex was repressed at this age. Immunofluorescent analyses and electron microscopy revealed a defect in laminin deposition and enlarged intercellular space in the Nmrk2-KO heart. The Nmrk2 gene is required to preserve cardiac function and structure during aging and becomes indispensable for maintaining NAD at late age. ...
http://europepmc.org...ct/med/29317476

 

First: that link doesn't lead to that abstract ... you appear to be quoting this thesis (click on the English version), which forms the basis for this paper, which we've discussed before.  IAC, again: they report a decline in NAMPT and NRK1 expression with age and  (and a reduction in NAMPT and an induction of NMRK2 expression in DCM), but NR had no effect on any of this (Fig. 7 I-K), and I see no reason to expect that it would. They "show that NR supplementation in food attenuates the development of HF in mice, more robustly in DCM, and partially after TAC, by stabilizing myocardial NAD⁺ levels in the failing heart," but the parsimonious explanation for that is that providing increased substrate allows for greater NMN (and thence NAD+) synthesis even at the same level and activity of NAMPT and NRKs.

[stefan_001]

 

Hi Michael, agree beyond the text I did not find they measured anything that would show upregulated transcription factor. I meant a systemic result of supplementation resulting in increase in NAMPT.

But then what is your basis for saying that supplementation resulted in an increase in NAMPT? They don't report any such thing, either at the protein, activity, or even the mRNA level, and there's no reason obvious to me to expect that it would do so: NAMPT's whole function is to produce NMN, and here they're bypassing its activity by supplying its product. That will raise NAD+ independently of any effect on NAMPT.
 

Here another report implicating decling NAMPT but also pointing to Nmrk1:
 
In different mice models of DCM, the expression of the nicotinamide riboside kinase 2 (Nmrk2) implicated in the synthesis of NAD,... is increased.. ...  NMRK2 was not essential to maintain myocardial NAD levels in response to pro-hypertrophic treatments and in young adults. However Nmrk1 and Nampt expression level declined strongly with aging and Nmrk2-KO mice displayed a 50% reduction in myocardial NAD levels at 24 months. The α7β1 integrin complex was repressed at this age. Immunofluorescent analyses and electron microscopy revealed a defect in laminin deposition and enlarged intercellular space in the Nmrk2-KO heart. The Nmrk2 gene is required to preserve cardiac function and structure during aging and becomes indispensable for maintaining NAD at late age. ...
http://europepmc.org...ct/med/29317476

 

First: that link doesn't lead to that abstract ... you appear to be quoting this thesis (click on the English version), which forms the basis for this paper, which we've discussed before.  IAC, again: they report a decline in NAMPT and NRK1 expression with age and  (and a reduction in NAMPT and an induction of NMRK2 expression in DCM), but NR had no effect on any of this (Fig. 7 I-K), and I see no reason to expect that it would. They "show that NR supplementation in food attenuates the development of HF in mice, more robustly in DCM, and partially after TAC, by stabilizing myocardial NAD⁺ levels in the failing heart," but the parsimonious explanation for that is that providing increased substrate allows for greater NMN (and thence NAD+) synthesis even at the same level and activity of NAMPT and NRKs.

 

 

Part 1: Didnt I write, agree cannot find it in the report? I think I am somewhat more speculative and hence look for different things. For example there are two NAD+-dependent proteins, SIRT1 deacetylase and PARP1 ADP-ribosyltransferase. These bind to CLOCK-BMAL1 and that in turn affects its DNA binding activity and target gene expression. Its also known that NAMPT is a direct transcriptional target of the CLOCK-BMAL1.So boosting NAD+ should/could have an influence on NAMPT. I would not see it impossibe there could be taking place a "systematic" shift towards a different balance in this feedback loop or another one under influence of continuous/ daily supplementation.

 

Part 2: the point was nmrk1 decline

 

[Michael]

Part 1: Didnt I write, agree cannot find it in the report? I think I am somewhat more speculative and hence look for different things. For example there are two NAD+-dependent proteins, SIRT1 deacetylase and PARP1 ADP-ribosyltransferase. These bind to CLOCK-BMAL1 and that in turn affects its DNA binding activity and target gene expression. Its also known that NAMPT is a direct transcriptional target of the CLOCK-BMAL1.So boosting NAD+ should/could have an influence on NAMPT. I would not see it impossibe there could be taking place a "systematic" shift towards a different balance in this feedback loop or another one under influence of continuous/ daily supplementation.

OK. But as I noted, the published DCM study (as vs. the original thesis paper) actually goes beyond not saying anything: they actively find there is no effect. Let that be a lesson to you not to invest so much brainpower in mechanistic speculation . IAC, for the reasons I gave, I don't think an hypothetical increase in NAMPT activity would make any sense. And if the elaborate metabolic Rube Goldberg device you posit actually happened, we'd all be in a perpetual state of feed-forward rises in NAMPT and NAD+ once we took our first NAD+-boosting supplement — like a metabolic perpetual motion machine with acceleration.

[stefan_001]

 

Part 1: Didnt I write, agree cannot find it in the report? I think I am somewhat more speculative and hence look for different things. For example there are two NAD+-dependent proteins, SIRT1 deacetylase and PARP1 ADP-ribosyltransferase. These bind to CLOCK-BMAL1 and that in turn affects its DNA binding activity and target gene expression. Its also known that NAMPT is a direct transcriptional target of the CLOCK-BMAL1.So boosting NAD+ should/could have an influence on NAMPT. I would not see it impossibe there could be taking place a "systematic" shift towards a different balance in this feedback loop or another one under influence of continuous/ daily supplementation.

OK. But as I noted, the published DCM study (as vs. the original thesis paper) actually goes beyond not saying anything: they actively find there is no effect. Let that be a lesson to you not to invest so much brainpower in mechanistic speculation . IAC, for the reasons I gave, I don't think an hypothetical increase in NAMPT activity would make any sense. And if the elaborate metabolic Rube Goldberg device you posit actually happened, we'd all be in a perpetual state of feed-forward rises in NAMPT and NAD+ once we took our first NAD+-boosting supplement — like a metabolic perpetual motion machine with acceleration.
 

 

you take things out of context by exaggeration, it would be illogical that there is no feedback loop that slightly reverses, halts or slows decline in NAMPT which develops over time.. Another example is the fact that miR-34a rises in obesity and inhibits NAMPT, NR research shows it works against obesity so logic is that there may be an effect on NAMPT as many people gain during ageing, simply carry more fat versus muscle. Or are you suggesting that if an miR-34a feedback loop would exist we should see that from taking the very first supplement, like obesity should dissapear in a day versus years?  When people do not explore possibilities you will never know they exist.

 

No worries about my brain, the investment of time in NR certainly has had a positive feedback loop on the aforementioned brain, health of myself, parents and finances.....quite the Rube Goldberg machine if you think of it.

Edited by stefan_001, 21 January 2018 - 08:50 AM.